- Shy, Brian R;
- Vykunta, Vivasvan S;
- Ha, Alvin;
- Talbot, Alexis;
- Roth, Theodore L;
- Nguyen, David N;
- Pfeifer, Wolfgang G;
- Chen, Yan Yi;
- Blaeschke, Franziska;
- Shifrut, Eric;
- Vedova, Shane;
- Mamedov, Murad R;
- Chung, Jing-Yi Jing;
- Li, Hong;
- Yu, Ruby;
- Wu, David;
- Wolf, Jeffrey;
- Martin, Thomas G;
- Castro, Carlos E;
- Ye, Lumeng;
- Esensten, Jonathan H;
- Eyquem, Justin;
- Marson, Alexander
Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.